THE FATTY ACID PATTERNS OF PLASMA
LIPIDS DURING ALIMENTARY LIPEMIA
V. P. Dole, … , M. A. Rizack, M. F. Sturman
J Clin Invest. 1959;38(9):1544-1554. https://doi.org/10.1172/JCI103933.
Research Article
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THE FATTY ACID PATTERNS OF PLASMA LIPIDS DURING
ALIMENTARY LIPEMIA*
By V. P. DOLE, A. T. JAMES, J. P. W. WEBB, M. A. RIZACK AND M. F. STURMAN t
(From The Rockefeller Institute, New York, N. Y., and the National Institute for Medical Research, London, England)
(Submittedfor publication March 11, 1959; accepted April 23, 1959)
The total amount of fatty acid contained in the blood stream at any moment is considerably less than the amount of fat that might be taken in a single feeding. Therefore, during the period of alimentary lipemia some of the lipids in blood would be expected to acquire the composition of dietary fat.
To illustrate, the blood plasma of a 70 Kg. man contains about 50 mEq. of fatty acids (approxi-mately 25 mEq. in phospholipids, 12 mEq. in cho-lesterol esters, 10 mEq. in triglycerides and 3 mEq. in nonesterified fatty acids), whereas a meal con-taining 3 ounces of fat provides 300 mEq. The amount of fatty acid entering the blood during a period of a few hours after such a feeding thus is about six times the total amount in circulation and, since the concentration of fatty acid in plasma remains approximately constant during absorption, the fatty acids of plasma must turn over on the average about six times. The actual turnover of transport lipids must be much greater than this since only a small part ofthe plasma lipid partici-pates directly inabsorption. Practically all of the newly absorbed fat enters the blood as triglyceride (1, 2). If dietary fat were simply mixed with this small pool and thentransported tothetissues,
the pool would turn over about 30 times and the composition of plasma triglyceride would become identical with that of dietary fat during the period of absorption.
Some evidence of this
expected
effect was ob-tained by Wilson and Hanner (3) who fed large doses ofcream orcod liver oilto children and ob-served that the iodine number of the increment of blood lipids during the lipemic phase became approximately equal to the iodine number of the fed fat. It is also known that the composition of*This research was supported in part by GrantA-2427
from the National Institutes of Health, United States Public Health Service, Bethesda, Md.
tFellow of the Dazian Foundation for Medical Research.
adipose tissue in various animals (4) and man
(5) can be changed by feeding diets containing large amounts of unsaturated acids; therefore, there can be little doubt that the blood stream must transport a substantial part of the dietary fattyacid
from gut to depot without modification.
On the other hand, a number of studies in the older literature [reviewed by Bloor (6)] showed that the iodine number and melting point of fat inthoracic duct lymph can differ significantly from thecorresponding values for the fat that had been
fed, and therefore that the fat delivered into the blood need nothavethe same fatty acid pattern as
the dietary mixture. More recently Fernandes,
Van de Kamer and Weijers (7) in studying a child with chylothorax found that the pattern of
fattyacids in thechyle was influenced bythe kind of fat fed during the preceding week, but signifi-cant differences between chyle and dietary fat
al-ways remained.
Inthe present study itwasfoundthatabsorption
of 100Gm. ofcornoil, coconutoilorbuttercaused
remarkably little change in thefatty acid patterns ofchylomicrainplasmaorinthe patterns of other plasma fractions. These results and the results of other studiesof the absorptive process must be
distinguished clearly from the effects of feeding some distinctive fat over a period of many weeks
(8-11). Inthe present caseone isconcerned with
a transport process; in the latter, with a slow
change in
composition
of tissue lipids caused byrepeated feedings and the manifestation of this
changeintheplasma offasting subjects.
METHODS
Chylomicra were separated from plasma by flotation into a supernatant layer of saline (100,000 GX30
min-utes) (12).
Nonesterified fatty acids were extracted into heptane
with theheptane-acid-isopropyl alcohol system previously
PLASMA FATTY ACIDS DURING ALIMENTARY LIPEMIA
extract, ethanol-water system with 0.02 N NaOH. After extraction of the alkaline ethanol-water phase with pe-troleum ether to remove contaminating esters, the
non-esterifiedfatty acidsweremethylated for chromatography. Plasma lipids were extracted with ether-ethanol; the extract was evaporated to dryness and the cholesterol ester plus triglyceride fraction separated from the phos-pholipids by extraction of the residue with acetone. The esterified fatty acids were saponified in 10 per cent methanolic KOH, extracted after removal of
nonsaponi-fiable material and methylated with methanolic HCl. Themethyl esters were storedassolutions inpetroleum
ether (B. P., 40 to 600 C.) in the cold until analyzed. Separationswerecarried outusingtwotypesofstationary
phase: a) Apiezon L vacuum stopcock grease at 1970 C. (14); and b) a polymer of ethylene glycol and adipic
acid at 175° C. (15-17). In the earlier work, the
gas-density balance (18) was usedas thevapor detector, total loads of ester separated being approximately 3 mg. At
this level of sensitivity large samples ofplasma were
re-quired (50 to 75 ml.) particularly whendealing with the nonesterified fatty acid fractions. The introduction ofthe much more sensitive argon ionization detector (19, 20) allowed a total column loadof100Ag. or less tobe used, so that more detailed physiological studies were possible with much smaller blood samples. Peak areas were meas-ured by triangulation and analyses were usually carried
out at two or more levels in order to determine major and minor components accurately. Theanalytical figures
given refer to percentage by weight of a given compo-nent in the acids of chain length C6 and C,.
The course of lipemia in some experiments was fol-lowed by determination of triglyceride by the method of Van Handel and Zilversmit (21).
EXPERIMENTAL PROCEDURE
Forcontrol data samples of plasma wereobtainedfrom twogroups ofhumansubjects: male medical students and a somewhat older group of male and female laboratory personnel. All subjects appeared healthy and of usual
dietary habits. At the time of testing all subjects were at least 12 hours post prandial. The analyses of animal plasma fractions were made on specimens obtained from
fasting animals maintained on commercial feeds.
Fasting male medical students from the blood donor list served as subjects for thefeeding experiments. After an initial sample of blood had been taken, each subject was fed 100 Gm. of test fat (corn oil, coconut oil or butter) with no other food. Thereafter they were bled three times: atone-half,one, and two and one-half hours (short-term studies) or at three, six and nine hours (longer
term). Duringthe period of observation the subjects re-mained ambulatory and were permitted water, ad libitun,
butnothingelse by mouth.
Bloodwas taken into heparinized flasks and centrifuged promptly to remove cells. Because of the relatively low
sensitivity of the chromatographic detector available in
1957 it was necessary to take about 100 ml. of blood in each sample. The more recent analyses have required
only 10 ml. of blood. Fortunately these later studies have given essentially the same results as the earlier ex-periments, which suggests that removal of 300 to 400 ml. of blood does not distort the fatty acid patterns, and so the data from the earlier and the more recent
experi-ments are considered together.
TREATMENT OF THE DATA
The wide variety of fatty acids now shown to occur in animal fats makes the familiar terminologyinadequate
and the systematic chemical nomenclature too cumber-some for tabulation ofanalytical results. We have found it convenient to identify fatty acids by a dual symbol:
the first figure denoting chain length, and the second,
the number of double bonds (e.g., 16: 0 for palmitic acid, 18: 1 for oleic acid and 18: 2 for linoleicacid). The symbol can be elaborated further to show whether the chain is branched or straight (e.g., branched 15: 0) and. positional isomers can be distinguished by suffixes (e.g.,
18:11o). However, even with this simplified tabulatioa it is difficult to compare mixtures having the same major but different minor components. We have explored t'he
possibility of using statistical methods for measuring Ihe
similarity of different patterns but the calculations h ave not been helpful to date because the relative importnce
ofdifferent components is not yet defined.
The "double bond index" (DBI) shown in the tables is a measure ofunsaturation, calculated from theformula,
DBI=Z(niwt/mi) (where n, is the number of {double
bonds in the fattyacid i, m, is the molecular weigohtand
w, the percentage by weight of the fatty acid in thegiven mixture). The factors ni/mi, given in Table I, were multiplied by the corresponding percentages obtained from the analysis and the products were summed to, give the number of double bonds per unit weight of mixed fatty acids.
RESULTS
Fasting subjects
Tables II and III give the fatty acid composition ofthe various plasma lipids isolated from both hu-mans and animals. The category "18: 1 isomers" refers to isomers of oleic acid that consist mainly oftrans-11:12 -octadecanoic acid (vaccenic)
(22);
20: 3 refers to an acid whose structure has beenpartially elucidated by degradation; and 18-22:?
referstoa group of unsaturated acids whose
struc-turehas yettobedetermined.
Short experiments
Table IV shows the compositions of the three different dietary fats and the fatty acid patterns
DOLE, JAMES, WEBB, RIZACK AND STURMAN
TABLE I
Numericalfactorsfor calculationofdoublebond index (DBI)
Acid 12:1 14:1 16:1 18:1 18:2 18:3 20:?
ni 0.505 0.442 0.394 0.354 0.714 1.080 1.315
mi
in the table being the average of two tests with Longer experiments
different subjects. Therewasessentiallynochange The
experiments running
up to nine hours didin the patterns except for a rise in lauric
(12:0)
sand myristic
(14:0)
acids after ingestion ofcoco-nut oil. Statistical analyses verified the signifi- and VI), but of relatively minor degree. For cance of these rises and also indicated that none instance, the chief components of corn oil feed-of the other variations in pattern (including fluc- ings, the unsaturated
C15
acids, comprised 89 per tuations in trace components not shown in the cent of the total load [oleic (18:1) and its isomers table) had any significant relation to the pattern accounted for 34 per cent, and linoleic (18:2) of dietary load. The double bond indices (which for about 55 per cent], yet during absorption,measure net unsaturation) remained essentially the
proportion
of 18:2 in eachplasma
fractionroseconstant.
only moderately,
remaining
well below thedietary
TABLE II
Composition of plasma lipidsin humansubjects*
Triglycerides-plus-Component NEFAt Chylomicra Phospholipid cholesterol-esters No.ofsubjects
Saturated
12:0 14:0 15:0 16:0
17:0
18:0 20:0
lonounsaturated
12:1 t4:1 6:1 3:19-10
i 1isomers
Diu~aturated
18t
Polyuisaturated 18:3
20:3
20A4
18
-.22
? SumDouble bond index
18
0.34:0.4
1.7:1 0.9
23.2 :4- 2.8
12.94--2.6
0.6 4: 1.0 2.4 4 0.9 28.9 :5.0}
3.7 -t 1. 3
14.5 4-4.1
1.34 1.5 4.7 41 2.6
94.2
28.0 i 5.7
6
1.9 : 1.5 3.7 i 1.4 0.8 : 0.8 24.2 :4: 4.7 0.4 41 0.4 10.44 1.7
0.9 I 1.0 1.4i:1.0 3.0 1: 1.0 21.84-4.8
8.7 4- 1.2 0.3 1 0.3 2.0 + 2.0 0.5 4 0.5 8.0 + 8.0
88.0
32.5 ± 11.4
6
0.1 0.1
1.1 0.8 0.5 1 0.5 33.1 13.8 0.5 : 0.5 12.8 :1 3.3 0.3 :1 0.4
0.441 0.1 0.5:4:0.5 2.5 :1: 0.7 18.64: 4.7
16.04: 6.5
0.5 ± 0.6 1.84 0.6 4.3 1: 2.1 4.1 4 4.0
97.1
33.3 4 3.5
6
0140
1.0 0.4 0.2 +0.2 21.44: 2.2 0.5 1: 0.3 4.2 4- 1.2
0 :1: 0.1 0.4 14 0.3 3.4-+ 1.0 26.441 5.5
33.5Az 5.5
0.5:1 0.5 1.041 0.5 4.2 41 2.0 2.0 4: 2.0
98.7
44.1 =1 2.8
*Values
givenin the main part of the table are the mean and standard deviation ofpercentageby weightfor each
component. Thesumisless than 100 per cent in each case because inconstant trace amounts ofunusualacids,detected
in individualanalyses,were omitted from the final tabulations. Thedouble bond index, definedinthe text,measures
degreeofunsaturation. tNonesterifiedfatty acids.
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PLASMA FATTY ACIDS DURING ALIMENTARY LIPEMIA
level, while 18:1 remained constant or fell so that the total C18 concentration remained essentially unchanged and the double bond index showed no
consistent trend.
The stability of the patterns is illustrated in
Figures 1 through 4, which show the fatty acid distributions classified by saturation and chain length. It will be seen in Figure 1 that chy-lomicra tended to maintain the pattern found in fasting samples, although the effect of the dietary load was perceptible. The pattern during ab-sorption, considered as a mixture between dietary fat and the body lipids with the fasting pattern, suggests that dietary fat contributed at most
only half of the oleic acid present in chylomicra (based on the elevation of 18:2) and, with respect to other components, a much smaller proportion.
The nonesterified fatty acids (Figure 2) also responded perceptibly to the dietary load, while
Food
Chylomicria
CoconuLt Thsting 3his. hr's. 9hrs.
:. oil
3216 1620
142 18:
Sotu- Umat- Uns7t-
Stu-Sated uated
ratedu'at-e
FIG. 1. PATTERNS OF FATTY ACIDS IN CHYLOMICRA AFTER FEEDING 100 GM. OF COCONUTOIL (UPPER Row)
OR CORN OIL (LOWER Row)
Thecomposition of dietary fatis shown ontheleft, fol-lowed by chylomicron patterns in blood samples obtained before feeding and at the indicated times subsequently. Saturated fatty acids are shown in solid black;
mono-unsaturated acids are shown with crosshatching, and
polyunsaturated, with stippling. The chain lengths, shown under thecorn oil diagram, apply similarly to each of the other diagrams.
Food
Coconut 50 oil
25
u is oil
soasC-u
25
12 16 16 20 1416 16
Sati&- UT-radeduroi
FIG. 2. N
NEFA
Satu- Moat*
rateduptk
TONESTERIFIED FATTY ACID (NEFA)
the patterns of triglyceride plus cholesterol esters (Figure 3) and phospholipids (Figure 4) were
virtually unchanged.
DISCUSSION
Fasting subjects
As has been noted before (23), each class of lipid was found to have a distinctive composition with a relatively small spread of values for major and minor components within each class (Table
II).
Thehigh concentration ofpolyunsaturated acids found in chylomicra is of particular interest since
chylomicra are known to contain less than 10
per cent of phospholipids (24, 25). It would seem, therefore, that either part of the trigly-cerideinchylomicra washighly unsaturated orthe
phospholipid component contained an
exception-ally large amount of unsaturated acids. In either
case some of the chylomicron lipid must have been derived from an endogenous source rich in
polyunsaturated acids. Inaddition the chylomicra
contained less linoleic acid than the other fractions. Some of the present data can be comparedwith those obtained in a previous study of British
DOLE, JAMES, WEBB, RIZACK AND STURMAN
75r Food
Coconut oil
50[
25
4.7 4) U
C4 4) 75
P4
Corn
50[
25[
Liii1A
1216 1620ULUILlLJII
1418 18
Satu-Unsot- Saw Unsat-ratedurted ratedurated
FIG. 3. TRIGLYCERIDES PLUS CHOLESTEROL ESTERS
similar in the two groups except that palmitic
acid appearsto be somewhat higher in the
Anmeri-can group and arachidonic acid, lower. Larger differences are seen in the cholesterol ester plus
triglyceride fraction; the American group had a
much higher linoleic level (33.5 + 5.5 per cent)
than the British (12.4 per cent), while the Brit-ish group showed a somewhat higher oleic acid level (34.7 per cent) than the Americans (26.4
+ 5.5 per cent). Presumably these differences
were due to higher cholesterol/triglyceride ratios in the American subjects.
Table III shows the fatty acid compositions of nonesterified fatty acids, phospholipds and chol-esterol esters plus triglyceride in plasma of sheep
cow, pig, horse and chicken. The three lipid
fractions appeared to have fairly consistent
pat-terns in all the animals studied; this similarity is
instrikingcontrast to the wide differences in kind
of food eaten by the various species.
Lipemia
The degree of alimentary lipemia depends both
on the kind of fat fed
(26)
and on the dose. Moderateloads, suchasmightbe eatenina normalmeal, usually cause little or no rise in total lipid concentration of plasma (27) although they are reflected in chylomicronemia and turbidity during the period of absorption (28). To obtain a sub-stantial rise in concentration of plasma fatty acids
one must feed a large amount of fat, such as 3.5
Gm.per Kg. (29). In the experiment of Wilson and Hanner (3) children were fed even larger doses, up to 5.6 Gm. per Kg. Changes in plasma fatty acid pattern did occur under these conditions, but in interpreting the results one must note the
unphysiological size of the load.
With the more moderate feedings used in the present experiments, the subjects in the corn oil group showed a slight rise in plasma triglyceride
concentration while those in the coconut oil group showed no significant change; all subjects, how-ever, showed definite turbidity of plasma during
absorption, usually maximal atsix hours.
Centri-fugation of the turbid plasma under a layer of saline in each case yielded a creamy chylomicron
layer and this, as is indicated in the tables, had a fatty acid composition quite different from the fed fat.
In interpreting the constancy of the fatty acid pattern of chylomicra it must be recognized that
some of the fatty acids in chylomicra are
con-tained in
phospholipids
and cholesterol esters(and so are not
necessarily
related to thedietary
Food Phospholipids
50 5
Coconult Fasting 3hi'z 6hr's. 9bra. oil
25 2
12 la
16?2
Sa~tu- I~- Sawe Uns rteCo urnt; rateduraM
FIG. 4. PHOSPHOLIPIDS
r.
load), and also that the flotation layer obtained from the upper part of the saline phase might be contaminated with nonchylomicron lipids. These explanations, however, fail to account for the discrepancy between chylomicra and fed fat. It
is unlikely that the phospholipids and cholesterol esters in chylomicra could have contributed more
than 10 per cent of the fatty acids (24, 25) and the composition of the chylomicron fraction was very far from what one would obtain by a mixture of nine parts of dietary fat with one part of phos-pholipid or any other fraction of the subnatant plasma. It also seems unlikely that any contami-nation of the flotation layer with nonchylomicron lipids could have been great enough to predomi-nate over the chylomicron lipids; centrifugation ofplasma fromfasting subjects yielded onlytraces
of lipids, while at the peak of lipemia, the same
procedure yielded a heavy cream. Finally, the
constancy in composition of the triglyceride plus cholesterol ester fraction showed that there could
not have been any appreciable proportion of un-modified dietary triglyceride in this fraction
(which includes chylomicron triglyceride) at the
time of any observation.
Two different kinds of process might account for the relative stability of the various plasma
fatty acid patterns during absorption. There
might be, as Bloor suggested (6), a conversion of
fatty acids during
digestion.
Alternatively, thefatty acid patterns of plasma lipids, including
chylomicra, might be held constant by rapid
re-cycling of fatty acids between blood and tissue
pools, as is the case with nonesterified fatty acids
(12, 30). Work now in progress in this labo-ratory indicates that the latter alternative is the
correct one.
Itis also of interest tonotethat the nonesterified fatty acid pattern did not follow changes in the
chylomicron pattern as might be expected if the
clearing of lipemia plasma were simply lipolytic
and the nonesterified fatty acids were products. Thisresultis in agreement with the experiments of Fredrickson, McCollester and Ono (31) who showed that not more than one molecule in 10 of the nonesterified fatty acids was derived from la-beled chylomicra when these were infused into dogs at rates comparable to that of normal ab-sorption.
SUMMARY
Plasma lipids were fractionated into chylomicra,
nonesterified fatty acids, triglycerides plus
chol-esterol esters and phospholipids, and the fatty acid compositions of these fractions weredetermined by
analysis with gas-liquid chromatography. The
data showed a fairly consistent pattern for each fraction in a group of normal fasting subjects. A few analyses made on animal plasmas yielded essentially the same patterns.
Plasmas taken during alimentary lipemia were analyzed similarly. It was found that neither the chylomicra, nor any other fraction, acquired the
pattern of dietary fat during the period oflipemia. This result suggests that the fatty acid pattern of
chylomicra in plasma is stabilized by equilibration with a larger pool of tissue lipids.
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